Establishment And Application Of A Specific PCR Method Based On The GRA14 Gene For Toxoplasma Gondii Detection

Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii,which is widely distributed throughout the world.Infection with T.gondii is very likely to cause clinical symptoms such as abortion and stillbirth in animals,which seriously threaten the healthy development of animal husbandry.Humans can also be infected by T.gondii,usually through eating a food containing T.gondii.Therefore,it is very important to monitor and control T.gondii for animal husbandry and food safety.Accurate diagnosis is necessary for the control of T.gondii.However,traditional pathogen examinations are time consuming and laborious,and the detection rate is low;serological methods such as indirect hemagglutination tests and enzyme-linked immunosorbent assays usually detect T.gondii antibodies and can be used to monitor the disease in a timely way.Polymerase chain reaction(PCR)has high accuracy,simple operation,and high specificity and sensitivity;it is better than conventional methods but requires primers with high specificity and sensitivity and suitable PCR conditions for the diagnosis of T.gondii.First,the present study used the T.gondii gene GRA14 as a target gene to establish a specific PCR for T.gondii detection.The results showed that the primers designed in this study have high specificity for T.gondii and can detect a minimum of 2.25 T.gondii tachyzoites.The optimal PCR and procedure is suitable for epidemiological investigation of T.gondii.An epidemiological survey of T.gondii in pigs from different provinces was conducted in 2016.The results showed that pig farms in Shanxi had the highest infection rate(31.7%),followed by Guizhou(27 %),Guangdong(13.4 %),and Fujian Province(8.9 %);nopositive samples were detected in Anhui Province.The epidemiological survey of T.gondii in the different growth stages of pigs showed that the infection rate of breeding pigs was high,with a rate of 18.6% in breeding sows and 12.7% in breeding boars.The infection rate of fattening pigs was lower(4.8%).Different kinds of samples,including blood,feed,urine,tissue,semen and water,were examined for the presence of T.gondii,of which the blood samples were most suitable for epidemiological surveys of T.gondii.The prevalence of T.gondii in the pork,beef and chicken from meat markets in Sihui city was investigated between May and November 2018.The results revealed that the infection rate of pork was the highest,at 25.00 %,followed by beef at 21.30 %.The infection rate of chickens was the lowest,at 15.74 %.Statistical analysis of T.gondii infection rates in different months showed that the infection rates of chicken and pork were highest in July,and that of beef was highest in November.In the present study,a specific PCR for T.gondii detection based on GRA14 was developed and used to investigate the prevalence of T.gondii infection in different pig farms from different provinces in China in 2016;1287 samples of pork,beef and chicken from different meat markets in Sihui city were also examined for the presence of T.gondii in 2018.These results provide information that will be helpful for the effective control of toxoplasmosis in animals and humans.